Should every embryo undergo preimplantation genetic testing for aneuploidy? A review of the modern approach to in vitro fertilization

Accepted Manuscript Should every embryo undergo preimplantation genetic testing for aneuploidy?: A review of the modern approach to in vitro fertilization Susan M. Maxwell, MD, James A. Grifo, MD, PhD PII:

S1521-6934(18)30163-9

DOI:

10.1016/j.bpobgyn.2018.07.005

Reference:

YBEOG 1842

To appear in:

Best Practice & Research Clinical Obstetrics & Gynaecology

Received Date: 12 February 2018 Accepted Date: 17 July 2018

Please cite this article as: Maxwell SM, Grifo JA, Should every embryo undergo preimplantation genetic testing for aneuploidy?: A review of the modern approach to in vitro fertilization, Best Practice & Research Clinical Obstetrics & Gynaecology (2018), doi: 10.1016/j.bpobgyn.2018.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Should every embryo undergo preimplantation genetic testing for aneuploidy?: A review of the modern approach to in vitro fertilization

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Susan M. Maxwell, MDa,b, and James A. Grifo, MD, PhDa. a

New York University Langone Fertility Center, 660 First Ave, 5th floor, NY, NY 10016.

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[email protected]. Corresponding author.

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Abstract

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Aneuploid conceptions constitute the majority of pregnancy failures in women of advanced maternal age. The best way to combat age related decline in fertility is through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A allows for better embryo selection which improves implantation rates with single embryo transfer and reduces miscarriage rates. Single embryo transfers decrease multiple gestations and adverse pregnancy outcomes such as preterm or low birth weight infants.

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Advancements in extended embryo culture, blastocyst biopsy techniques, and 24-chromosome aneuploidy screening platforms have made PGT-A safe and accessible for all patients undergoing in vitro fertilization. Improved genomic coverage of new sequencing platforms, such as next generation

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sequencing, have increased the identification and diagnosis of mosaicism and partial aneuploidies in preimplantation embryos. Mosaic embryos have decreased viability compared to euploid embryos

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when transferred, but some mosaic embryos result in normal live births. Whole genome amplification artifacts may contribute to a misdiagnosis of mosaicism or some mosaic embryos may self-correct to euploid after implantation. For this reason, patients without euploid embryos should be given the option of transferring mosaic embryos after genetic counseling. Further research is needed to characterize which mosaic embryos may be viable.

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Key Words: Preimplantation genetic testing for aneuploidy, embryonic mosaicism, next generation

INTRODUCTION

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quantitative polymerase chain reaction.

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sequencing, array comparative genomic hybridization, single nucleotide polymorphism array,

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Preimplantation genetic testing (PGT) is the process by which a preimplantation embryo undergoes biopsy of either a blastomere at the cleavage stage (Day-3) or trophectoderm (TE) cells at the blastocyst stage (Day 5-7). The biopsied specimens then undergo genetic analysis with various sequencing platforms. There are 3 main applications for the use of this technology. Preimplantation genetic testing for aneuploidy (PGT-A) involves biopsy specimens undergoing 24-chromosome screening for aneuploidy. Embryos are then diagnosed as either euploid with a normal compliment of chromosomes, aneuploid with an abnormal compliment of chromosomes, partial aneuploid with one portion of a chromosome

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missing or duplicated in all cells, mosaic containing 2 different cell lines within the same embryo (often one euploid cell line and one aneuploid cell line), or partial mosaic with one euploid cell line and one partial aneuploid cell line. Preimplantation genetic testing can also be performed to select unaffected

chromosomal structural rearrangements (PGT-SR)(1).

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embryos in patients who are carriers of monogenic/single gene defects (PGT-M) or those with

PGT-A is utilized for patients with recurrent pregnancy loss or implantation failures due to age related

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decline in fertility. Harton et al 2013 (2), demonstrated that implantation rates remain stable across all age groups if euploid embryos are transferred; therefore, aneuploidy is the predominate cause of age

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related decline in fertility. PGT-A is increasingly being offered to all patients undergoing in vitro fertilization (IVF) regardless of age or infertility diagnosis due to its advantages over conventional IVF. PGT-A allows for the selection of embryos with the best potential for survival and normal development which increases implantation rates and reduces miscarriage rates (2). Increased implantation rates with

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PGT-A have paved the way for single embryo transfer, significantly reducing multiple gestations(3). Fewer twin and higher order multiple pregnancies results in fewer preterm and low birth rate infants. Spontaneous abortions and the need to terminate abnormal gestations are emotionally distressing for

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patients undergoing infertility treatments. These complications can be minimized using PGT-A and healthy pregnancies can be achieved faster(4). Cost remains a significant limiting factor for the use of

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PGT-A in all IVF patients, and controversies surrounding the accuracy of PGT-A diagnostic platforms have hampered its universal use. The purpose of this review is to describe the technological advancements that have contributed to modern PGT-A, the current controversies surrounding the technology, and limitations of PGT-A.

HISTORY OF PREIMPLANTATION GENETIC TESTING

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Pre-implantation genetic testing emerged in the early 1990’s as a means to select female embryos for transfer in carriers of recessive x-linked disorders (5, 6). Shortly thereafter, this technology was expanded to identify embryos carrying a single gene disorder, such as cystic fibrosis (7). Bastomere

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biopsies were performed at the cleavage stage and specific probes were used in polymerase chain reactions (PCR) to detect affected embryos. Fluorescent in situ hybridization (FISH) on blastomere biopsies was another technique used as a means of sex selection for carriers of recessive x-linked

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disorders, but it provided the additional benefit of detecting chromosomal copy number(8, 9). FISH probes were developed in order to detect the copy number of the most common aneuploidies which

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involved chromosomes X, Y, 18, 13, and 21(10).

Fertility rates were known to decrease with increasing maternal age, and women undergoing IVF over age 40yo were observed to have lower implantation rates and higher miscarriage rates. This was initially thought to be due to uterine aging, but studies of donor oocyte cycles suggested that oocyte

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aging was the cause. Sauer et al 1992 (11), compared women 40 yo or older undergoing oocyte donation compared to women under 40 years old undergoing oocyte donation for premature ovarian insufficiency and women 40 yo or older undergoing IVF using autologous oocytes. Oocyte donation

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cycles resulted in improved implantation and ongoing pregnancy rates irrespective of recipient age compared to women over 40yo using autologous oocytes. Age related decline in fertility disappeared

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with the use of donor oocytes(11). This, however, did not explain the mechanism behind why oocyte competence decreased with increasing maternal age. Cytogenetic analysis of products of conception demonstrated that around 50% of spontaneous abortions were chromosomally abnormal, and a chromosomally abnormal conceptus was associated with increased maternal age (12). Munné and colleges were similarly finding high rates of aneuploidy among human pre-implantation embryos(10). They studied monospermic cleavage stage arrested embryos and normally developed surplus embryos from women over 40 years old using FISH for

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chromosomes X, Y, 18, 13, and 21. Chromosomal abnormalities were identified in 70% of arrested embryos (14/20) and 70% of morphologically normal cleavage-stage embryos in women over 40 years old (7/10)(10). Chromosomally abnormal conceptions were therefore contributing to age related

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decline in fertility. PGT-A using FISH on blastomere biopsies of cleavage-stage embryos was proposed as a treatment for women with implantation failures after in vitro fertilization (IVF) or recurrent pregnancy loss (13).

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Early PGT-A randomized controlled trials (RTCs) did not show a benefit and showed possible harm with the procedure. Mastenbroek et al 2011 (14), published a systemic review and meta-analysis which

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included nine randomized controlled trials on PGS. Eight of the nine RTCs performed cleavage-stage biopsy of 1-2 blastomeres. One of the RTCs performed TE biopsy of blastocysts. All of the included RTCs used FISH for chromosomes X, Y, 18, 13, and 21. On average 1.5-2 embryos were transferred in each study. Women of advanced maternal age undergoing PGT-A with FISH had a significantly lower live birth

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rate than those undergoing IVF without PGT-A (18% vs 23%, risk difference: -0.08; 95% CI -0.13 to -0.03). There was no difference between miscarriage rates or multiple pregnancy rates in women of advanced maternal age(14).

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Many technical limitations contributed to the failure of these early PGT-A trials to show improved pregnancy outcomes. The diagnostic efficiency of FISH is low with 5-20% of biopsied embryos failing to

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have a diagnosis. FISH is only able to detect aneuploidy involving the chromosomes tested, and the accuracy per probe is 92-99% resulting in a high overall error rate (14). Cleavage-stage embryos have high rates of embryonic mosaicism, and mosaicism is not identified with a single blastomere biopsy(1518). Mosaicism rates decrease with extended embryo culture, suggesting that embryos self-correct or euploid cell lines predominate at later developmental stages(19). Therefore, an aneuploidy diagnosis resulting from a single blastomere, would exclude a potentially viable embryo from transfer.

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Extended embryo culture to the blastocyst stage improved embryo selection and made embryo biopsy safer. Adler et al 2014 (20), compared euploidy rates between patients undergoing cleavage-stage blastomere biopsy and blastocyst-stage TE biopsy. They found that the average total number of euploid

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embryos per retrieval cycle were the same between groups, but the percentage of euploid embryos were higher in the blastocyst biopsy group than in the cleavage-stage biopsy group (42% vs 24%).

Extended embryo culture helped to select for euploid embryos and was not detrimental to normal

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embryo development(20). Scott et al 2013 (21), performed a paired RTC comparing cleavage-stage single blastomere biopsy and transfer with no biopsy and blastocyst-stage TE biopsy and transfer with

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no biopsy. Cleavage-stage blastomere biopsy was associated with a 39.1% relative reduction in implantation rate compared to blastocyst-stage TE biopsy with a non-significant relative reduction of 3%(21). Culture to the blastocyst stage could be safely applied to all patients and blastocyst-stage TE biopsy allowed for more robust genetic testing of embryos.

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Advances in embryo cryopreservation techniques have streamlined the use of PGT-A by allowing patients to freeze all embryos while awaiting genetic testing results. The most updated Cochrane Review on fresh embryo transfer versus a freeze-all strategy with subsequent frozen embryo transfer

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showed moderate evidence that there was no difference in cumulative live birth rates between treatment groups(22). When comparing the two embryo cryopreservation methods, vitrification results

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in improved embryo survival rates over slow freezing, but there is no difference in pregnancy outcomes after frozen embryo transfer between the methods(23). Cryopreservation of all embryos followed by frozen embryo transfer may have several advantages over fresh embryo transfer including reduced risk of ovarian hyperstimulation syndrome (22), improved endometrial receptivity in high responders (24), reduced ectopic pregnancy rates (25), and improved obstetric and perinatal outcomes(26). These advancements in extended embryo culture, embryo cryopreservation, and embryo biopsy techniques have influenced the way IVF is practiced today. The modern approach to IVF using PGT-A

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involves culturing embryos to day 5 or 6 (blastocyst stage) and then performing a TE biopsy of approximately 5-10 cells. Rush PGT-A can be performed through certain labs with results within 24 hours allowing for a fresh embryo transfer; however, most centers vitrify all biopsied embryos. Patients

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return several weeks later after receiving PGT-A results for a single thawed euploid embryo transfer (STEET). Differences in modern PGT-A practices are mainly due to the genetic platform used to process TE biopsies.

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METHODS FOR PGT-A

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Many different methods are available for 24-chromosome screening for PGT-A, and they include array comparative genomic hybridization (aCGH), single nucleotide polymorphism array (SNP array), quantitative PCR (qPCR), and next-generation sequencing (high and low resolution) (NGS). All of these techniques are designed as screening tests and have been extensively validated for detecting aneuploidy. They differ depending on their genomic coverage and ability to detect unbalanced

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translocations, partial aneuploidies, polyploidy, and mosaicism. Array CGH, SNP array, and high resolution NGS utilize whole genome amplification (WGA) of genomic DNA which can introduce artifact or an inability to produce results from a specimen. Quantitative PCR and low resolution NGS do not

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utilize WGA; therefore, they have lower genomic coverage and are unable to detect small deletions and duplications. All of the platforms are capable of performing simultaneous detection of single gene

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defects if specific probes are created beforehand (27-32). It is important to understand the differences between these technologies and the limitations of each when interpreting PGT-A results. Array CGH has been extensively validated for PGT-A in the literature (4, 33). Amplified genomic DNA along with reference DNA are fluorescently labeled with distinct colors. Selected DNA probes (approximately 4000) which are representative of conserved sequences on each chromosome are annealed to the microarray. The genomic DNA and reference DNA then compete for hybridization on

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the microarray. The fluorescent signal from the hybridized DNA is detected by a computer, and software calculates the chromosomal copy number of the genomic DNA compared to the reference DNA which is known to have a normal compliment of chromosomes(34). In addition to aneuploidy, aCGH can detect

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unbalanced translocations, partial aneuploidies (5-20 Mb), and mosaicism if over 40-60% of biopsied cells are abnormal (27, 35-37). Array CGH, however, cannot reliably detect polyploidy which occurs in 12% of conceptions(38). The estimated rate of discrepant diagnoses for PGT-A with aCGH is 1-2% (39).

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Single nucleotide polymorphisms (SNPs) are positions within the genome where one of two nucleotides exist. These are known as alleles and are labeled A or B. These alleles are variable within the population,

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and each person has two copies of each allele (one on each chromosome). With SNP array fluorescently labeled probes bind to approximately 300,000 SNPs spaced throughout the genome. Different colors are given off depending on the nucleotide present at the SNP. A genotype is assigned to each SNP location (AA, AB, or BB) and this is compared to a reference genome (27, 40). In addition to aneuploidy,

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SNP array can detect unbalanced translocations, partial aneuploidies, triploidy, and uniparental disomy, but reporting of mosaicism is limited. Aneuploidy results are also not available if consanguinity exists (27). The reported error rate is 2-4%, with errors from embryos diagnosed as aneuploid resulting in a

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chromosomally normal live birth (29, 41).

Quantitative PCR is a rapid technique for determining embryo aneuploidy that provides results within 4

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hours allowing for fresh embryo transfer. Multiplex PCR amplification is performed on 96 selected loci spread throughout the genome (4 per chromosome). Relative quantification of the 4 loci on each chromosome is performed relative to a reference gene on that chromosome. Then the chromosomal copy number can be calculated based on ΔΔCt values for that chromosome (27, 28, 42). Quantitative PCR has limited detection of triploidy, and does not detect unbalanced translocations, partial aneuploidies, or mosaicism. The error rate of qPCR is approximately 1% (28).

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Next-generation sequencing is the newest platform for 24-chromosome aneuploidy screening providing the highest genomic coverage. Genomic DNA undergoes WGA, fragmentation, and then library preparation where adapters and barcodes are added. For the MiSeq platform from Illumina, a bridge

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PCR step is performed followed by optics-based sequencing by synthesis. During this process primers anneal to template DNA and a fluorescent signal is given off as nucleotides are added. With the

Personal Genome Machine (PGM) from Thermo-Fisher Scientific an emulsion PCR step is performed.

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During DNA synthesis hydrogen ions are released as nucleotides are added causing a shift in pH which is detected by sensors. With high resolution NGS whole genome sequencing is performed at a depth of

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1X. Sequenced data is compared to a reference genome to determine chromosomal copy number. High resolution NGS is able to detect unbalanced translocations, partial aneuploidies (1.8-14 Mb), triploidy, and mosaicism with 20% or more abnormal cells in the biopsy (43, 44). The clinical error rate of NGS is estimated to be between 1-2% (45).

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Low resolution, low density, or targeted NGS differs from high resolution NGS in that it does not involve WGA. Approximately 10,000 loci spread throughout the genome are selectively amplified. Then NGS is performed as above. Low resolution NGS is designed to only detect whole chromosome aneuploidy as it

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cannot detect abnormalities between amplified loci(27). Zimmerman et al 2018 (31), has developed a method of targeted NGS that involves multiplex amplification of genomic DNA using 96 unique primers.

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This approach increases the depth of sequencing compared to high resolution NGS allowing for simultaneous genotype analysis. Genotyping aids in the detection of triploidy and allows for DNA fingerprinting (31). Among aneuploid embryo re-biopsy studies, targeted NGS had a concordance rate for detecting aneuploidy of 98.7% per embryo compared to qPCR (31). LIMITATIONS OF CURRENT PGT-A TECHNOLOGIES

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With improvements in sequencing technology, abnormalities such as partial aneuploidy, mosaicism, and partial mosaicism have been increasingly reported on PGT-A results. This has decreased the number of embryos available for transfer in IVF cycles (46). Although error rates with all methods of 24-

mosaicism, or partial mosaicism are unknown (47).

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chromosome aneuploidy detection are low, clinical error rates with diagnoses of partial aneuploidy,

Cell line mixing studies have been performed to validate the detection of whole and partial chromosome

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mosaicism with NGS compared to aCGH (48, 49). Studies comparing results between aCGH and NGS on clinical samples using the same WGA product do not rule out the chance that the mosaicism or partial

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aneuploidy was due to artifact introduced during amplification (47). Embryo re-biopsy studies have been performed in mosaic embryos showing that that the reproducibility of a TE biopsy demonstrating mosaicism is only 41-58% (43, 50, 51). A TE biopsy of 5 cells may not be representative of the degree of mosaicism of the entire embryo (47).

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There are a few studies that have sought to validate the detection of partial aneuploidies with NGS. Vera-Rodriguez et al 2016 (52), studied the concordance of a diagnosis of partial aneuploidy and partial mosaicism between aCGH and NGS using the same WGA product from a TE biopsy. Validation of the

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diagnosis of partial aneuploidy was performed on a select number of the blastocysts using FISH. Concordance between all three methods was 92.9% (26/28 blastocysts)(52). A re-biopsy study was

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recently performed on 26 blastocysts with small chromosome deletions using VeriSeq NGS(53). The original diagnosis was detected in 24/26 (92.3%) embryos on re-biopsy, and the deletion was present in both the inner cell mass and TE in 14/26 embryos (53.9%)(53). Larger studies are needed to confirm these findings.

The limits of detection of mosaicism for NGS is approximately 20% as determined by various mixing studies of euploid and aneuploid cell lines (43, 48, 54). Some studies have questioned the reliability of

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protocols set to detect mosaicism at this low level. Goodrich et al 2016 (55), performed blinded cell line mixing studies and compared the detection of mosaicism between VeriSeq NGS on the MiSeq platform and qPCR. Using factory settings for aneuploidy analysis (detection of 50% mosaicism or more), there

level of 20-80%, the false positive rate was increased to 33%(55).

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were no false positive results. However, when custom settings were used to detect mosaicism at the

Each genomics lab has a different cut off for the reporting of mosaicism. For example, some labs report

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mosaicism if 20-80% of cells in the biopsy specimen are aneuploid, and others report mosaicism of 3070% only. Mosaics above or below the cut off would be called aneuploid or euploid. Some labs do not

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report mosaicism at all with mosaicism of >40-50% considered anuploid and mosaicism below the cut off euploid. Practitioners should know what the cut offs are for the genomics lab they are using and if mosaicism is reported. If mosaicism is not reported, then potentially viable embryos could be deemed aneuploid and never considered for transfer (48). Inconsistencies in the diagnosis of mosaicism and the

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difficulty in validating platforms for the detection of mosaicism has created controversies over whether mosaicism should be reported.

Proponents for the reporting of mosaicism argue that increased screening and reporting of mosaicism

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with NGS has resulted in improved pregnancy outcomes. Friedenthal et al 2018 (46), found that ongoing pregnancy rates after single euploid embryo transfer significantly increased from 54.4% to 62% with the

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switch of PGT-A from aCGH to NGS. A multicenter study of the clinical outcomes of 143 mosaic embryo transfers diagnosed by NGS documented an implantation rate of 53%, an ongoing pregnancy rate of 41%, and a spontaneous abortion rate of 24% versus 71%, 63%, and 10% for euploid embryo transfers (48). Reanalysis of WGA products from euploid embryos diagnosed with aCGH that resulted in miscarriage found that 31.6% were mosaic with NGS, suggesting that mosaicism could be a cause of miscarriage in the first trimester(49). Mosaic embryos may represent a third category of embryos that are potentially viable but with diminished developmental potential.

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More clinics are choosing to transfer mosaic embryos after extensive genetic counseling if patients do not have euploid embryos available for transfer and it is not possible for the patient to undergo another IVF cycle. Deciding which mosaic embryo to transfer can be challenging. The Preimplantation Genetic

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Diagnosis International Society (PGDIS) published a position statement about which mosaic embryos should be preferentially transferred. They recommend against transferring mosaics resulting in known syndromes in the literature such as trisomies involving chromosomes 21, 18, 13, and 22. Autosomal

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monosomies should be selected over trisomies because monosomic cell lines are not viable (except monosomy X); however, mosaicism due to mitotic non-disjunction can result in both monosomic and

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trisomic cell lines within an embryo that may be undetected. Mosaicism caused by a trisomy or monosomy rescue event leads to uniparental disomy which can cause various syndromes if chromosomes 7, 14, and 15 are involved. Confined placental mosaicism of chromosomes 2, 7, 16, and 22 may result in IUGR or an increased risk of fetal demise (56).

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Romana Grati et al 2018 (57), investigated the likelihood of mosaicism detected at chorionic villus sampling (CVS) being present in the fetus at amniocentesis. Mosaicism was detected in 2.1% of CVS cases and aneuploidy was confirmed in 13.3% of those undergoing amniocentesis. Based on the

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likelihood of fetal involvement, each mosaic chromosome was assigned a risk score. Mosaics which should not be transferred due to a high risk of fetal involvement and/or risk of fetal syndromes are

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trisomies 13, 14, 16, 18, 21, and 45, X. Those with the lowest risk with transfer (composite score of 0) are mosaic trisomies 1, 3, 10, 12, and 19. Those with a composite score of 1 are mosaic trisomies 4 and 5 and 47, XYY. A composite score of 2 was assigned to mosaic trisomies 2, 7, 11, 17, and 22, a composite score of 3 to mosaic trosomies 6, 9, and 15, and a composite score of 4-5 to mosaic trisomies 8, 20, 47, XXX, and 47, XXY. This scoring system will assist in counseling patients about the risk of miscarriage or having an affected fetus with the transfer of mosaic embryos(57).

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There is no absolutely safe mosaic embryo to transfer; however, most of the viable mosaic embryos have resulted in chromosomally normal infants. Aneuploid cell lines within a mosaic embryo may preferentially persist in the TE and undergo apoptosis in the inner cell mass resulting in confined

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placental mosaicism and a normal fetus(58). Patients choosing to transfer mosaic embryos should therefore undergo prenatal testing. It is important to note that confined placental mosaicism could

for a definitive diagnosis of the fetus (59).

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RANDOMIZED CONTROL TRIALS FOR 24-CHROMOSOME PGT-A

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result in a diagnosis of aneuploidy with cell-free DNA testing or CVS. Amniocentesis may be necessary

Modern approaches to PGT-A have resulted in significantly higher pregnancy rates compared to conventional IVF without PGT-A. Several randomized control trials have documented a benefit with the use of PGT-A, even among good prognosis patients. PGT-A helps women achieve pregnancy faster by selecting the embryo most likely to implant and result in a live birth. Miscarriage rates and terminations

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due to aneuploid fetuses are decreased with PGT-A. Most importantly, single embryo transfers are possible with PGT-A, reducing twin gestations, and decreasing preterm and low birth weight infants. Scott et al 2013 (60), performed a RCT comparing pregnancy rates between patients undergoing PGT-A

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versus conventional IVF. Patients were randomized into either the study or control groups if they had 2

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or more blastocysts available. Patients in the control group underwent a fresh embryo transfer on day 5. Patients in the study group underwent PGT-A with qPCR followed by fresh embryo transfer on day 6. Patients in both groups were good prognosis patients with an average age of 32yo. The average number of embryos transferred in the PGT-A group was 1.86 and in the control group 2. The implantation rate was significantly higher in the PGT-A group at 79.8% versus 63.2% for the control group (p=0.002). The ongoing pregnancy rate was also significantly higher in the PGT-A group at 66.4% compared to 47.9% in the control group (p=0.001)(60).

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Forman et al 2013 (3), performed another RCT comparing PGT-A with single embryo transfer to conventional IVF with double embryo transfer. PGT-A was performed using qPCR. Patient were randomized if they had 2 or more blastocysts available. The mean age was 34-45 years old for each

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group. For the intention to treat analysis, the ongoing pregnancy rate in the PGT-A group was 60.7% compared to 65.1% in the control group (non-significant with 95% CI -18.7%-9.9%). The rate of multiple gestations was significantly higher in the control group (53.4% vs 0%, p<0.001). Among women over

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35yo, the sustained implantation rate was significantly higher in the PGT-A group compared to the control group (58.3% vs 39.8%, p=0.03). The miscarriage rate was 11.5% in the PGT-A group compared

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to 20% for the control group, which was not statistically significant(3).

Yang et al 2012 (61), performed a RCT comparing single embryo transfers with PGT-A using aCGH versus conventional IVF in good prognosis patients <35 years old. Fresh embryo transfers were performed in both groups on day 6. The average age of patients in both groups was 31-32 years old. The clinical

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pregnancy rate was significantly higher in the PGT-A group at 70.9% compared to 45.8% in the control group (p=0.017), and the ongoing pregnancy rates were 69.1% for the PGT-A group versus 41.7% for the control group (p=0.009). The miscarriage rate was 2.9% in the PGT-A group and 9.1% in the control

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group, this was not statistically significant (p=0.597)(61). These RCTs of good prognosis patients have shown a benefit to PGT-A. Trials including poor prognosis

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patients are harder to initiate because patients with diminished ovarian reserve may not have blastocysts available for biopsy or they may not have euploid embryos available for transfer after PGT-A. Critics of PGT-A argue that poor prognosis patients do not benefit from aneuploidy screening for these reasons, and they would be better served by having a fresh transfer of multiple embryos. Retrospective studies of PGT-A in patients over 35yo have demonstrated a clear benefit, but these include only patients with euploid embryos available for transfer. Maxwell et al 2017 (62), performed a

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retrospective study comparing cumulative pregnancy rates between patients undergoing PGT-A with NGS and those undergoing conventional IVF. The study included patients in the PGT-A group who did not have euploid embryos for transfer as part of an intention to treat analysis. After multiple embryo

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transfers, there were no significant differences in ongoing pregnancy rates per retrieval across any age group. This is further evidence that a significant number of embryos are not damaged or excluded due to PGT-A. Furthermore, the cumulative miscarriage rate per clinical pregnancy was significantly higher

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in women ages 38-42 undergoing conventional IVF compared to those using PGT-A (28.6-46.4% versus 11.7-14.7%) (62). Prospective studies of cumulative pregnancy rates per retrieval cycle using PGT-A or

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conventional IVF would be helpful to evaluate the best treatment for poor prognosis patients. SUMMARY

Advances in embryo culture techniques, embryo biopsy, and PGT-A technology have resulted in improved implantation and pregnancy rates across all age groups. PGT-A can be safely offered to all

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patients as a way to improve implantation rates with single embryo transfer, reducing miscarriage and multiple pregnancies. New genetic platforms for PGT-A are more sensitive at picking up chromosomal abnormalities such as mosaicism and partial aneuploidies which contribute to implantation failures and

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miscarriage (48, 49). PGT-A is a screening test, therefore, there is a possibility of false positives and negatives, especially with subtle chromosomal abnormalities such as mosaicism(55). For this reason,

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mosaic embryos should be considered for transfer if no euploid embryos are available. More studies are needed to test the limits of detection of the various PGT-A platforms and the reliability of diagnoses of mosaicism, partial aneuploidy, and partial mosaicism. Studies of the viability of mosaic embryos need to be expanded, and determining which mosaic embryos are more favorable for transfer than others is necessary. Genetic counseling is necessary for patients both before and after PGT-A testing. Patients must understand the limits of current PGT-A technologies in addition to the benefits.

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PRACTICE POINTS •

Age-related decline in fertility is predominately due to an increased rate of aneuploid

•

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conceptions in women over age 35yo. Preimplantation Genetic Testing for Aneuploidy is a way to screen preimplantation embryos for all 24-chromosomes. Embryos with a normal compliment of chromosomes are transferred increasing implantation rates and decreasing miscarriage rates.

New genetic platforms for PGT-A, such as next generation sequencing, have higher genomic

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•

coverage allowing for the detection of mosaicism, partial aneuploidy, and partial mosaicism. Mosaic embryos have decreased implantation rates and increased miscarriage rates, but some

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are capable of resulting in a live birth of a chromosomally normal infant. RESEARCH AGENDA •

Prospective, blinded, non-selection studies are needed to determine the clinical error rates of

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embryos diagnosed as mosaic, partial aneuploidy, and partial mosaic with next generation sequencing.

Studies investigating the viability of mosaic embryos.

•

Prospective studies of cumulative pregnancy rates per retrieval cycle using PGT-A or

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•

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conventional IVF to evaluate the best treatment for poor prognosis patients. ACKNOWLEDGEMENTS None

Conflicts of interest

The authors have no conflicts of interest.

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PRACTICE POINTS •

Age-related decline in fertility is predominately due to an increased rate of aneuploid

•

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conceptions in women over age 35yo. Preimplantation Genetic Testing for Aneuploidy is a way to screen preimplantation embryos for all 24-chromosomes. Embryos with a normal compliment of chromosomes are transferred increasing implantation rates and decreasing miscarriage rates.

New genetic platforms for PGT-A, such as next generation sequencing, have higher genomic

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•

coverage allowing for the detection of mosaicism, partial aneuploidy, and partial mosaicism. Mosaic embryos have decreased implantation rates and increased miscarriage rates, but some

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•

are capable of resulting in a live birth of a chromosomally normal infant.

•

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RESEARCH AGENDA

Prospective, blinded, non-selection studies are needed to determine the clinical error rates of embryos diagnosed as mosaic, partial aneuploidy, and partial mosaic with next generation sequencing.

Studies investigating the viability of mosaic embryos.

•

Prospective studies of cumulative pregnancy rates per retrieval cycle using PGT-A or

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conventional IVF to evaluate the best treatment for poor prognosis patients.

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Highlights • •

Preimplantation genetic testing for aneuploidy (PGT-A) involves biopsy specimens undergoing 24-chromosome screening for aneuploidy PGT-A allows for the selection of embryos with the best potential for survival and normal development which increases implantation rates and reduces miscarriage rates Early PGT-A utilized blastomere biopsy at the cleaveage stage with FISH for chromosomes 13, 18, 21, X, and Y.

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Early PGT-A randomized controlled trials (RTCs) did not show a benefit and showed possible harm with blastomere biopsy.

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FISH had a low diagnostic efficiency and could only identify aneuploidy involving chromosomes 13, 18, 21, X and Y.

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Embryo cryopreservation techniques have improved, and recent studies do not see a difference in cumulative live birth rates between fresh embryo transfers and frozen embryo transfers.

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The modern approach to IVF involves extended embryo culture to the blastocyst stage, trophectoderm biopsy of 5-10 cells, preimplantation genetic testing for aneuploidy of all 24 chromosomes, and single euploid embryo transfer.

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The current methods available for 24-chromosome PGT-A are aCGH, SNP array, qPCR, and NGS (high and low resolution).

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All genetic platforms are validated for the detection of whole chromosome aneuploidy, but they differ in their detection of unbalanced translocations, partial aneuploidies, polyploidy, and mosaicism.

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High resolution next generation sequencing has the highest genomic coverage allowing for the detection of unbalanced translocations, partial aneuploidies (1.8-14 Mb), triploidy, and mosaicism with 20% or more abnormal cells in the biopsy.

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Increased detection of mosaicism has decreased the number of embryos available for transfer.

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Although error rates with all methods of 24-chromosome aneuploidy detection are low, clinical error rates with diagnoses of partial aneuploidy, mosaicism, or partial mosaicism are unknown.

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Whole genome amplification of genomic DNA can introduce artifacts that may appear as mosaicism. Not all genomics labs report mosaicism, and different labs use different cut offs for the detection of mosaicism.

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The reporting of mosaicism may be helpful because mosaic embryos have decreased implantation rates and increased miscarriage rates.

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Mosaic embryos should be considered for transfer if there are no euploid embryos available because some mosaic embryos can result in a chromosomally normal live birth.

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Mosaic embryos involving trisomies of chromosomes 21, 18, 13, and possibly 22, 14, 16 or monosomy X should not be transferred due to their associating with known syndromes.

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Mosaicism involving chromosomes 7, 14, and 15 can cause uniparental disomy.

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Confined placental mosaicism of chromosomes 2, 7, 16, and 22 can cause IUGR and risk of fetal demise.

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Patients undergoing transfer of mosaic embryos should strongly consider prenatal testing with amniocentesis.

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Several RTCs have shown increased implantation rates with PGT-A in good prognosis patients.

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PGT-A allows for single embryo transfer which significantly reduces the rate of multiple gestations.

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RCTs including poor prognosis patients are needed comparing PGT-A to conventional IVF.

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Studies of the viability of mosaic embryos need to be expanded.

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